Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy

Journal: Redox Report : Communications in Free Radical Research

Article Title: Ferroptosis inhibition and mitochondrial rescue: a novel mechanism of emodin in rheumatoid arthritis

doi: 10.1080/13510002.2026.2646383

Figure Lengend Snippet: EMO improves joint histopathological changes and structural damage in CIA mice. (A) EMO's chemical structure. (B) Schematic of the animal experiment. (C) Representative photographs of the hind paws of mice in each group. (D) Representative H&E staining images of ankle joints in each group (200×); red arrow: indicates inflammatory cell infiltration. (E) Representative Safranin O–Fast Green staining images of ankle joints in each group (100×, 200×); blue arrow: indicates cartilage erosion or bone destruction. (F) Representative Micro-CT images of the hind paws of mice in each group; yellow arrows: indicates cartilage erosion or bone destruction. (G–I) Quantitative analyses of Tb.Th, BV/TV and BMD in the hind paw bones of mice (n = 6); data are presented as mean ± standard deviation (SD). ### p < 0.001 vs control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs the model group. Control group: normal mouse group; Model group: CIA mouse group; EMO-L group: low-dose EMO group, 25 mg/kg; EMO-M group: medium-dose EMO group, 50 mg/kg; EMO-H group: high-dose EMO group, 100 mg/kg; MTX group: methotrexate group, 10 mg/kg; H&E: hematoxylin and eosin staining; TEM: transmission electron microscopy; Tb.Th: trabecular thickness; BV/TV: bone volume/total volume; BMD: bone mineral density.

Article Snippet: Specimens of mouse paws were preserved in 4% paraformaldehyde for a duration of 24 h and subsequently examined with a high-resolution micro-CT system (Skyscan1276, Bruker Corporation, USA).

Techniques: Staining, Micro-CT, Standard Deviation, Control, Transmission Assay, Electron Microscopy

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibit RANKL-induced osteoclastogenesis. A The chemical structural formula of Tetrandrine; B Tetrandrine impact on BMMs viability was detected by CCK-8 assay at 48 h and 96 h. C With different dilutions of Tetrandrine, M-CSF and RANKL for 7 days, BMMs were processed; scale bar = 200 μm. D The number and area of TRAP-positive cells. E TRAP-positive BMMs after the treatment with 1 μM Tetrandrine for the designated days in the process of osteoclastogenesis; scale bar = 200 μm. F To quantify TRAP-positive cells, the number and area of osteoclasts. G Immunofluorescence image for showing the formation of F-actin. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: CCK-8 Assay, Immunofluorescence, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits osteoclastic bone resportion and expression of osteoclastogenesis related genes. A BMMs were planted into slices of bone and treated with different concentrations of Tetrandrine for 7 days and bone resorption pit pictures taken by a scanning electron microscope are presented; scale bar = 500 μm. The pit zone resorption area were shown by the ImageJ. B The detection of expression levels of osteoclastogenesis related genes in BMMs with 0, 0.25, 0.5, 1 μM was carried out with qPCR. C After treating BMMs with Tetrandrine (1 μM) for 0, 1, 3, 5 days, they were detected via RT-qPCR. The mRNA expression levels of NFATc1, c-Fos, DC-STAMP, TRAP, CTSK were evaluated. D For 0, 1, 3, 5 days with or without 1 μM of Tetrandrine, the treatment of BMMs was carried out with RANKL. The protein expression levels of NFATc1, TRAP and CTSK were evaluated. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine affects osteoclastogenesis by promoting the ubiquitination of TRAIL. A The mRNA expression level of TRAIL in BMMs with 0, 0.25, 0.5, 1 μM of Tetrandrine was carried out with RT-qPCR. B The expression of level of TRAIL in BMMs treated with specified concentrations of Tetrandrine for 5 days was detected using western blot. C Immunofluorescence image for showing TRAIL expression in cytoplasm; scale bar = 200 μm. D The co-IP assay was used to detect the effect of Tetrandrine on TRAIL ubiquitination with or without RANKL. E–G The transfection efficiency of TRAIL silencing (si #1, si #2, si #3) and TRAIL overexpression (TRAIL vector) detected via RT-qPCR and western blotting. H–K BMMs were transfected with siTRAIL and TRAIL vector, the expression of the related genes and proteins that were revealed by RT-qPCR and western blotting. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Co-Immunoprecipitation Assay, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: TRAIL overexpression partially rescued the inhibitory effect of Tetrandrine on osteoclastogenesis. A The mRNA expression of c-Fos, CTSK, DC-STAMP, NFATc1 and TRAP in BMMs in rescue experiment was detected by RT-qPCR. B Western blotting demonstrated the expression of NFATc1, TRAP and CTSK in BMMs in rescue experiment. C The number and area of TRAP-positive cells, the bone resorption pits and F-actin formation in rescue experiment; Scale bar in TRAP staining and immunofluorescence image = 200 μm; scale bar in bone resorption pit picture = 500 μm. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits NF-κB and MAPK pathways by downregulating TRAIL expression. A Treatment of BMMs was carried out with RANKL, with or without 0.25 μM Tetrandrine for 0, 30 and 60 min. The p-P65, p-JNK, p-ERK, p-P38, p-IκBα, p-IKKα/β protein levels were evaluated. B The p-P65, p-JNK, p-P38, p-IκBα, p-IKKα/β protein levels were determined by western blotting in rescue experiment. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine promotes C3H10 osteoblastogenesis and inhibits TRAIL expression in C3H10. A Effect of Tetrandrine on C3H10 viability by CCK-8 assay at 48 h and 96 h. B The TRAIL, OCN, ALP and OPG expression levels in C3H10 treated with Tetrandrine were detected with RT-qPCR. C The protein levels of COL1a, RUNX2, OCN, TRAIL, RANKL and OPG were measured with western blotting. D , E ALP and ARS expression in C3H10 following the treatment with various concentration of Tetrandrine for 7 days. The mineralized extracellular matrix was detected with Alizarin Red S staining for 14 days. The OD values obtained for ALP solutions and mineralized matrix solutions after their treatment with Tetrandrine; scale bar = 200 μm. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Staining, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine prevents OVX-induced bone loss in vivo. A Weight change diagram of mice after Tetrandrine treatment (each; n = 5). B RANKL/OPG, TNFα and IL-6 levels in serum of mice were determined with Elisa assay. C , D Detection of the tetrandrine effects on the expression of TRAIL in the OVX-induced model carried out with RT-qPCR and western blot. E Detection of NFATc1, DC-STAMP, TRAP and CTSK expression levels was carried out with RT-qPCR. F Calcein-alizarin red S labeling to detect the femur mineral apposition rate (MAR) in mice. G TRAP staining was used for sections of femurs and number of osteoclasts per field of each specimen were measured. H The Von kossa staining was used for the sections of femurs and the MS/BS (%) was measured. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Staining, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits OVX-induced osteoporosis in mice model. A H&E staining was used for several major organs of mice in three groups (each; n = 5). B , C The micro-CT was used for the scanning tibias of each mouse, and the BS/BV, Tb.N, Tb.Sp and Tb.Th values were recorded for each specimen. D , E H&E and Masson staining was also used for the femurs. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05 and ** P < 0.01 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Staining, Micro-CT, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Schematic illustration for the mechanism by which Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL. RANKL recruits TRAF6 by interacting with its receptor RANK, and then activates downstream effectors such as NF-κB and MAPKs to promote mature osteoclast formation. Inflammatory factors including IL-6 and TNFα play an active role in the binding of RANK and RANKL and Tetrandrine has obvious anti-inflammatory effect. More importantly, Tetrandrine promotes TRAIL degradation and TRAIL downregulation inhibits the recruitment of TRAF6 by RANKL and eventually dampen osteoclast different and function. Besides, Tetrandrine can stimulate osteoblast difference

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Antibodies used for western blot, immunoperoxidase immunostaining, immunofluorescence and co-immunoprecipitation analyses.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Western Blot, Immunostaining, Immunofluorescence, Immunohistochemistry

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Average fold changes of Slc26 gene family members during maturation stage relative to secretory stage based on genome-wide mRNA transcriptome analysis. (N/A, expression not detected; α = 0.05).

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Genome Wide, Expressing

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: The expression levels of Slc26a1, Slc26a6 and Slc26a7 were normalized to those of Beta-Actin and are presented in fold changes. The expression levels of Slc26a1, Slc26a6 and Slc26a7 were up-regulated by ~10.0, ~4.1 and ~15.3 fold, respectively, at maturation stage relative to secretory stage.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Expressing

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: A1-C1. Protein-level expression of Slc26a1, Slc26a6 and Slc26a7 was detected by western blot analysis using samples obtained from both secretory- and maturation-stage enamel organs (4-week-old rat incisors). Protein samples extracted from kidney (4-week-old rat) were used as reference controls. The molecular weights for Slc26a1, Slc26a6 and Slc26a7 are 75kDa, 90kDa and 72kDa, respectively. Beta-Actin served as the control for sample loading. A2-C2. The intensities of the bands (relative to Beta-Actin) were measured using ImageJ. The average fold changes of Slc26a1, Slc26a6 and Slc26a7 at the protein level were ~1.6, ~6.1, ~4.2, respectively.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Immunostaining procedures were applied to the sagittal sections prepared from paraffin-embedded 4-week-old rat mandibles. A. Slc26a1 in secretory-stage ameloblasts (S); B. Slc26a1 in smooth-ended ameloblasts at maturation stage (M-SA); C. Slc26a1 in ruffle-ended ameloblasts at maturation stage (M-RA); D. Slc26a6 in secretory-stage ameloblasts (S); E. Slc26a6 in smooth-ended ameloblasts at maturation stage (M-SA); F. Slc26a6 in ruffle-ended ameloblasts at maturation stage (M-RA); G. Slc26a7 in secretory-stage ameloblasts (S); H. Slc26a7 in smooth-ended ameloblasts at maturation stage (M-SA); I. Slc26a7 in ruffle-ended ameloblasts at maturation stage (M-RA); J-L. The sections that were incubated without antibodies served as negative controls for immunostaining. All images were collected under 20x magnification. Scale bar shown in Panel J (50μm). Slc26a1, Slc26a6 and Slc26a7 all showed expression on the apical membrane and/or within subapical cytoplasmic region (double black arrows). Positive staining in other regions was indicated by double black asterisks. SI—Stratum intermedium; Am—Ameloblast; ES—Enamel space; CT—Connective tissue; PL—Papillary layer.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Immunostaining, Incubation, Expressing, Membrane, Staining

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: (A-B) Co-localization of Slc26a1 with Ae2 and Lamp1 in maturation-stage ameloblasts by confocal microscopy at 63x magnification. The signals of Slc26a1 were mainly seen on the apical membrane of maturation-stage ameloblasts (Panels A and B; Green). In contrast, Ae2 was localized to the basolateral membrane (Panel A; Red) and the basal pole (Panel C; Red), while Lamp1 showed a cytoplasmic and/or peri-nuclear distribution pattern in ameloblasts (Panel B; Red). The sections were stained with DAPI to highlight the nuclei (Panels A and B; Blue). (C-D) Co-localization of Slc26a6 with Ae2 and Lamp1 in maturation-stage ameloblasts. Slc26a6 exhibited a similar expression pattern to that of Slc26a1—on the apical membrane of maturation-stage ameloblasts (Panels C and D; Green). The fluorescence signals of Ae2 (Panel C; Red) and Lamp1 (Panel D; Red) were used as references. The sections were stained with DAPI to highlight the nuclei (Panels C and D; Blue). (E-G) Co-localization of Slc26a7 with Ae2, Lamp1 and Cftr in maturation-stage ameloblasts. The expression of Slc26a7 was found both on the apical membrane and within the cytoplasmic region (Panels E-G; Green). The fluorescence signals of Slc26a7 partially overlapped with those of Lamp1 (Panel F; Red) and Cftr (Panel G; Red), rather than Ae2 (Panel E; Red). The sections were stained with DAPI to highlight the nuclei (Panels E-G; Blue). The images were collected under confocal microscopy (63x magnification). Scale bar shown in Panel G (10μm).

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Confocal Microscopy, Membrane, Staining, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Co-IP was conducted using protein samples extracted from the maturation-stage enamel organs of 4-week-old rat incisors (50~100 μg initial input). The interaction complexes were pulled down by anti-Cftr antibody . The subsequent western blot analyses were performed using primary antibodies to Slc26a1, Slc26a6 and Slc26a7 . The positive control was the pre-cleared total protein without the following immunoprecipitation procedures, and the negative control was prepared by skipping the step of applying the antibody against Cftr.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Positive Control, Immunoprecipitation, Negative Control

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: The mandibles from wild-type, Slc26a1 null and Slc26a7 null animals (at 8 weeks of age) were subject to micro-CT analysis (n = 3). The relative density and thickness of enamel on the labial incisor where the cortical bone enclosing just begins (A1-C2) were measured. There was no statistical difference between mutant and wild-type animals with respect to these two parameters (D-E). (Enamel Em, Dentin De)

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Micro-CT, Mutagenesis

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: The surface of the enamel of incisor and molars in Slc26a1 -/- and Slc26a7 -/- animals were similar to those of wild-type animals (A1-B3, magnification 35x). When the internal structures of enamel (incisor) were observed in coronal section (C1-C3, magnification 5000x), the enamel from mutant animals showed a mild disruption in rod density and diameter (C2-C3) compared with wild-type enamel (C1). The arrangement of the enamel rod and inter-rod structures (incisor) did not seem to be impacted significantly by the deletion of Slc26a1 or Slc26a7 (C1-C3).

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Mutagenesis, Disruption

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Enamel and dentin Vickers microhardness of mutant animals.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Mutagenesis, Control

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: (A-C) EDS spectrum of mature enamel in wild-type, Slc26a1 -/- and Slc26a7 -/- animals (n = 6 per group). (D) No statistically significant differences were detected in the At% of Ca, P and O between mutant and wild-type enamel. (E) The At% of Cl increased significantly by ~34% in both Slc26a1 -/- ( P = 0.012) and Slc26a7 -/- ( P = 0.035) enamel. There was a significant decrease in the At% of C in Slc26a1 -/- enamel ( P = 0.028). A similar difference in the At% of C was detected between Slc26a7 -/- and wild-type enamel, but was only marginally significant ( P = 0.078). The At% of Na in Slc26a7 -/- enamel also significantly decreased ( P = 0.028).

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Most genes that showed significant changes in expression were up-regulated (Panels A-S), indicating a compensatory effect induced by the deletion of Slc26a1 or Slc26a7. The expression values of differentially expressed genes were normalized to those of Beta-Actin . For all the two-tailed t tests used, the significance level was 0.05. * <0.05; ** <0.01.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: SLC26A Gene Family Participate in pH Regulation during Enamel Maturation

doi: 10.1371/journal.pone.0144703

Figure Lengend Snippet: Cftr, Slc26a1, Slc26a4, Slc26a6 and Slc26a7 are localized to the apical membrane and Slc26a1, Slc26a6 and Slc26a7 physically interact with Cftr to form regulation complexes. Slc26a7 is also found on the endo-lysosomal membrane. Ae2, Nhe1 and NBCe1 are localized basolaterally. Clcn7 is expressed on the endo-lysosomal membrane. CA2 exhibits intracellular distribution whereas CA6 functions in extracellular enamel matrix. Lamp1 in this image was used as a marker of late lysosomes.

Article Snippet: Proteintech (10708-1-AP) , Slc26a1 (rat) , immunohistochemistry (dilution 1:300).

Techniques: Membrane, Marker

Journal: Journal of Translational Medicine

Article Title: A novel molecule ZYZ329 targeting histone methyltransferase SMYD3 suppresses pathological angiogenesis via the driven mitochondrial ROS/HIF-1α/VEGFA axis

doi: 10.1186/s12967-025-07580-2

Figure Lengend Snippet: SMYD3 inhibition attenuated angiogenesis in vitro and in vivo. ( A ) HUVEC were transduced with lentiviral ShRNA (ShRNA1 and ShRNA2) or control ShRNA (Sh-Ctrl) for 48 hours. SMYD3 and H3K4me3 protein expression was quantified by Western blot, with GAPDH and total histone H3 serving as internal reference ( n = 3). ( B ) The VEGF-containing Matrigel plugs with Sh-SMYD3 or Sh-Ctrl were harvested from WT mice after 14 days ( n = 8). Bar = 3 mm. ( C, D ) Sections of Matrigel plugs were stained with H&E or CD31 (green) and DAPI (blue). Representative images and quantification of CD31 positive areas were shown ( n = 8). Bar = 100 μm. ( E ) HUVEC were incubated with Sh-SMYD3 and then seeded on Matrigel; tube formation was captured (upper panel) and relative tube length were analyzed (lower panel). n = 6. Bar = 100 μm. ( F ) the thoracic aortic ring isolated from WT mice were cultured in VEGF-containing Matrigel with Sh-SMYD3 or Sh-Ctrl. Sprouting was captured (lower panel) and sprouting/explant ratio was calculated (upper panel). n = 8. Bar = 100 μm. ( G ) The CAM was photographed after incubating with Sh-SMYD3 or Sh-Ctrl ( n = 6). Wound healing assay ( H ), cell counts ( I ), and nitric oxide ( J ) were measured ( n = 6). Bar = 100 μm. Unpaired Student’s t test ( D, E, F, H, J ), One-way ANOVA ( A ) and Two-way ANOVA ( I ) coupled with Bonferroni post hoc test were used. ns p > 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: After mounting in antifade mounting medium with DAPI (Beyotime), the slides were observed and photographed under a confocal microscope (SpinSR10, Olympus).

Techniques: Inhibition, In Vitro, In Vivo, Transduction, shRNA, Control, Expressing, Western Blot, Staining, Incubation, Isolation, Cell Culture, Wound Healing Assay

Journal: Journal of Translational Medicine

Article Title: A novel molecule ZYZ329 targeting histone methyltransferase SMYD3 suppresses pathological angiogenesis via the driven mitochondrial ROS/HIF-1α/VEGFA axis

doi: 10.1186/s12967-025-07580-2

Figure Lengend Snippet: Overexpression of SMYD3 promoted angiogenesis. ( A ) HUVEC were transfected with Sh-Ctrl, Sh-SMYD3, and Sh-SMYD3 plus SMYD3-Res lentiviruses for 48 hours. SMYD3 and β-tubulin protein expression was quantified by Western blot ( n = 3). ( B ) The VEGF-containing Matrigel plugs with Sh-Ctrl, Sh-SMYD3, and Sh-SMYD3 plus SMYD3-Res lentiviruses were harvested from WT mice after 14 days ( n = 6). Bar = 4 mm. ( C ) Sections of Matrigel plugs were stained with CD31 (green) and DAPI (blue). Representative images and quantification of CD31 positive areas were shown ( n = 6). Bar = 100 μm. ( D ) HUVEC were incubated with Sh-Ctrl, Sh-SMYD3, and Sh-SMYD3 plus SMYD3-Res and then seeded on Matrigel; tube formation was captured and relative tube length was analyzed ( n = 5). Bar = 100 μm. ( E ) Matrigel plugs and ( F ) CD31 immunofluorescence staining ( n = 6). Bar = 100 μm. ( G ) Aortic ring sprouting ( n = 5). Bar = 100 μm. ( H ) Tube formation stained with Calcein AM (green) ( n = 5). Bar = 100 μm. Data shown are for Ad-SMYD3 and Ad-Ctrl groups. Unpaired Student’s t test ( F, G, H ), One-way ANOVA ( A, C, D ) coupled with Bonferroni post hoc test were used. ** p < 0.01, *** p < 0.001

Article Snippet: After mounting in antifade mounting medium with DAPI (Beyotime), the slides were observed and photographed under a confocal microscope (SpinSR10, Olympus).

Techniques: Over Expression, Transfection, Expressing, Western Blot, Staining, Incubation, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: A novel molecule ZYZ329 targeting histone methyltransferase SMYD3 suppresses pathological angiogenesis via the driven mitochondrial ROS/HIF-1α/VEGFA axis

doi: 10.1186/s12967-025-07580-2

Figure Lengend Snippet: ZYZ329 significantly attenuated the angiogenic ability of endothelial cells. Matrigel plugs containing 10 μM ZYZ329, EPZ031686, or DMSO were sectioned and stained with H&E for histological analysis ( A ) and CD31 (green)/DAPI (blue) for immunofluorescence detection ( B ). Bar = 100 μm. Representative images were shown, and the CD31-positive area was quantified ( D , n = 6). Aortic ring sprouting area was photographed ( C ) and quantified as sprouting/explant ratio ( E , n = 6). Bar = 100 μm. Endothelial tube formation in Matrigel was captured ( G ), and the corresponding relative tube length was quantified ( F , n = 6). Bar = 100 μm. ( H ) The wound closure ratio was analyzed ( n = 6). ( I ) HUVEC proliferation was assessed by cell counting on day 0 and day 3 post-treatment with 10 μM ZYZ329, EPZ031686, or DMSO ( n = 6). ( J ) Measurement of NO production in HUVEC stimulated by VEGF after treatment with 10 μM ZYZ329, EPZ031686, or DMSO ( n = 6). Matrigel plugs ( K ) and corresponding CD31 staining ( L ) following treatment with 1 µM, 5 µM, and 10 µM ZYZ329 ( n = 6). ( K ) Bar = 4 mm. ( L ) Bar = 100 μm. ( M ) Aortic ring assay under treatment with 1 µM, 5 µM, and 10 µM ZYZ329 ( n = 5). Bar = 100 μm. One-way ANOVA ( D, E, F, H, J, L, M ) and Two-way ANOVA ( I ) coupled with Bonferroni post hoc test were used. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: After mounting in antifade mounting medium with DAPI (Beyotime), the slides were observed and photographed under a confocal microscope (SpinSR10, Olympus).

Techniques: Staining, Immunofluorescence, Cell Counting, Aortic Ring Assay

Journal: Journal of Translational Medicine

Article Title: A novel molecule ZYZ329 targeting histone methyltransferase SMYD3 suppresses pathological angiogenesis via the driven mitochondrial ROS/HIF-1α/VEGFA axis

doi: 10.1186/s12967-025-07580-2

Figure Lengend Snippet: ZYZ329 inhibited pathological angiogenesis in vivo. ( A ) Establishment and monitoring of murine hindlimb ischemia model. Schematic in this panel was created using BioRender (https://www.biorender.com/). ( B, C ) Vehicle, ZYZ329, and EPZ031686 were administered via intraperitoneal injection (25 mg/kg) for 14 consecutive days. The Sham group underwent skin incision without femoral artery ligation. Hindlimb perfusion was quantitatively assessed using laser speckle imaging, with ischemic/non-ischemic hindlimb perfusion ratio calculated pre-ligation and at postoperative days 0, 3, 7, and 14 ( n = 8). ( D ) High resolution laser speckle imaging was employed to characterize vascular network architecture in ischemic hindlimbs across experimental groups: Sham, Vehicle, ZYZ329, and EPZ031686 ( n = 6). ( E ) On postoperative day 14, micro-CT angiography was performed following injection of angiographic contrast agent, enabling three-dimensional reconstruction and analysis of collateral vessel distribution in ischemic hindlimbs ( n = 6). ( F ) H&E staining of ischemic hindlimb muscles in the Sham, Vehicle, ZYZ329, and EPZ031686 groups. Myofibers with central nuclei in the injured/regenerating areas are indicated by arrows ( n = 8). Bar = 50 μm. ( G ) Representative images of immunofluorescence staining of CD31 (green) and DAPI (blue) in ischemic hindlimb muscles in the Sham, Vehicle, ZYZ329, and EPZ031686 groups ( n = 8). Bar = 50 μm. ( H ) Percentage quantification of the injured/regenerating areas in ischemic hindlimb sections ( n = 8). ( I ) Analysis of CD31 staining fluorescence intensity in Sham, Vehicle, ZYZ329, and EPZ031686 groups ( n = 8). Two-way ANOVA ( C ) and One-way ANOVA ( H, I ) coupled with Bonferroni post hoc test were used. ** p < 0.01, *** p < 0.001

Article Snippet: After mounting in antifade mounting medium with DAPI (Beyotime), the slides were observed and photographed under a confocal microscope (SpinSR10, Olympus).

Techniques: In Vivo, Injection, Ligation, Imaging, Micro-CT, Staining, Muscles, Immunofluorescence, Fluorescence

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: The combination of Tegavivint and panobinostat exerts synergistic anti-MM effects in vitro and ex vivo. ( A ) Seven HMCL were treated with clinically relevant doses of panobinostat (up to 20 nmol/L) for 48 h and PI+ was measured by FC ( n = 3–6). Cell viability was calculated as a relative percentage compared with vehicle alone (100%). U266 were further treated with increasing doses of panobinostat up to 200 nmol/L for 48 h and propidium iodide + (PI+) was measured by flow cytometry (FC) ( n = 6). ( B ) NCIH929, OCIMy1, and U266 were treated with Tegavivint, panobinsotat, or their combination for 48 h. PI+ was measured by FC ( n = 3). The expected drug combination responses were calculated based on Bliss reference model using SynergyFinder. Deviations between observed and expected responses with positive and negative values denote synergy and antagonism respectively. ( C ) Non-malignant HS5 cells were treated with similar doses of Tegavivint or panobinostat alone for 48 h and PI+ was measured by FC. Cell viability was calculated as a relative percentage compared with vehicle alone (100%) ( n = 3) (left panel). HS5 cells were treated with Tegavivint, panobinsotat, or their combination for 48 h and PI+ was measured by flow cytometry (FC) ( n = 3). Dose response matrix was produced (right panel). ( D ) Proportion of apoptotic (Apo 2.7 +) CD38 + CD45 − primary MM cells in an autologous bone marrow coculture assay after 72 h of Tegavivint, panobinostat, or the combination. Numbers represent calculated Bliss synergy scores for each patient sample.

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: In Vitro, Ex Vivo, Flow Cytometry, Produced, Co-culture Assay

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: The combination of Tegavivint and panobinostat induces apoptotic cell death and down-regulation of β-catenin targets. ( A ) Percentage of annexin V, annexin V/7AAD, and 7AAD-positive cells after Tegavivint, panobinostat or the combination for OCIMy1 and U266 at 24 and 48 h respectively ( n = 3, ±SE). 2way ANOVA Dunett’s multiple comparisons test of row means to control row mean (DMSO treated) (* p = 0.0175, ** p = 0.0013, *** p = 0.0007, **** p < 0.0001 for OCIMy1 and * p = 0.0164, **** p < 0.0001 for U266). ( B ) Percentage of active caspase-9 after treatment with Tegavivint, panobinostat, or the combination for OCIMy1 and U266 at 24 and 48 h respectively relative to the vehicle (DMSO) treated cells ( n = 3, ±SE). ( C ) Immunoblotting of whole-cell lysates for β-catenin down-stream targets Myc, Cyclin D1 and Cyclin D2 after 24 or 48 h of treatment with Tegavivint, panobinostat, or the combination for OCiMy1 and U266. Loading control: β-actin ( n = 3). Detailed information about the Western blotting can be found at .

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: Control, Western Blot

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: Tegavivint in combination with panobinostat targets mitochondrial fitness of MM cells. ( A ) OCIMy1 and U266 cells were treated with Tegavivint (Teg-), panobinostat (Pan-), Tegavivint and panobinostat, or vehicle (DMSO) for 20 h. Mitochondrial transmembrane potential was measured by TMRE staining and FC. MFI was measured and expressed as a relative % to the TMRE MFI of UT (untreated) cells (UT = 100%) ( n = 3, ±SE, nonparametric Kruskal-Wallis multiple comparisons test, ** p = 0.0013, *** p = 0.001 for OCIMy1, and * p = 0.04 and 0.03 for U266 respectively). ( B ) OCIMy1 and U266 were treated with Tegavivint, panobinostat or the combination for 20 h and stained with TMRE (red for mitochondrial potential), Mito Tracker Green (green for mitochondria) and Hoechst 33,342 (blue for nuclei) and evaluated with confocal microscopy. One μmol/L of the uncoupling agent FCCP was added 20 min before imaging and used as a positive control (magnification 40× WI).

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: Staining, Confocal Microscopy, Imaging, Positive Control

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: Tegavivint in combination with panobinostat reduces oxidative phosphorylation of MM cells. ( A ) OCIMy1 and U266 cells were treated with vehicle, 100 nmol/L of Tegavivint, 10 nmol/L of panobinostat, or the combination for 18 h. Analysis of ECAR (extracellular acidification rate) was performed using a Seahorse XF analyser to assess glycolysis in the context of a glycolysis stress test. Three technical replicates were performed for each experimental variable ( n = 3, ±SE) (Glu: glucose, OM: oligomycin, 2DG: 2-deoxy-D-glucose). ( B ) OCIMy1 and U266 cells were treated similarly to A for 18 h. Analysis of OCR (oxygen consumption rate) was performed using a Seahorse XF analyser to assess mitochondrial respiration/oxphos (oxidative phosphorylation) in the context of a mitochondrial stress test. Three technical replicates were performed for each experimental variable ( n = 3, ±SE) (OM: oligomycin, FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone R/A: rotenone/antimycin). ( C ) Results from the OCR analysis were further analysed and the basal respiration, maximal respiration, and ATP production for OCIMy1 and U266 were calculated ( n = 3, ±SE) (nonparametric Kruskal–Wallis multiple comparisons test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001).

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: Phospho-proteomics

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: Tegavivint overcomes acquired panobinostat resistance in bortezomib resistant MM cells. The HMCL KMS12PE was cultured for a 12 months period in slowly increasing concentrations of bortezomib (VR), carfilzomib (KR), or vehicle (parental). Resistant cells were stored for 6 months, thawed and cultured for an additional 12 week period in the absence of drugs to confirm the acquisition of permanent drug resistance. ( A ). Cells were treated with increasing doses of bortezomib or carfilzomib for 48 h and cell viability was calculated after PI staining and FC (% of vehicle treated cells). ( B ). Parental, VR and KR cells were treated with increasing doses of panobinostat for 48 h and cell viability was calculated after PI staining and FC (% of UT) (left panel). Parental, VR and KR cells were treated with increasing doses of Tegavivint for 48 h and cell viability was calculated after PI staining and FC (% of UT) (right panel). For calculations of LD 50 we applied the non-linear regression model. Dashed lines correspond to the non-linear fit curves. ( C ). Parental and VR cells were treated with Tegavivint, panobinostat or the combination for 48 h and the PI+ was measured by FC ( n = 3). Dose response matrices were produced for both cell lines and the expected drug combination responses were calculated based on the Bliss reference model using SynergyFinder. Deviations between observed and expected responses with positive and negative values denote synergy and antagonism respectively.

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: Cell Culture, Staining, Produced

Journal: Cancers

Article Title: Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

doi: 10.3390/cancers14030840

Figure Lengend Snippet: Tegavivint in combination with panobinostat exerts potent anti-MM effect in vivo, without a negative impact on bone metabolism. ( A ) NSG human MM-bearing mice received vehicle (17% Solutol HS 15 (Sigma-Aldrich) and normal saline) ( n = 5), whereas treated mice received 10 mg/kg panobinostat ( n = 5) or 30 mg/kg Tegavivint ( n = 5), or their combination ( n = 5) twice a week intraperitoneally for 3 consecutive weeks for 2 cycles of 28 days each. Tumour growth was monitored weekly with in vivo bioluminescence imaging. Dorsal (D) and ventral (V) views of NSG human MM-bearing mice at day 49 (end of cycle 2 treatment). The median flux (photons/second) of dorsal and ventral views for each cohort is documented ( n = 5 for each cohort, ±SD). Multiple t -tests were used for statistical analysis without assuming SD, and statistical significance was determined using the Holm-Sidak method, with α = 5.000%. ↓: therapeutic intervention. ( B ) Adult NSG human MM-bearing mice were treated as mentioned above for two cycles. Upon reaching experimental end point mice were humanely euthanised and micro-CT of L5 vertebrae was performed. BoneJ was used for the analysis and the calculation of bone morphometric indices and compared with the measurements derived from three healthy age matched NSG mice. Treatment with single drugs or the combination did not negatively affect the bone volume fraction (BV/TV), trabecular thickness (Tb. Thickness) or the connectivity density (Conn. D) (* p ≤ 0.05, nonparametric Mann-Whitney t test). ( C ) During the last week of cycle 2 serum was analysed for osteocalcin levels by ELISA. Derived values were compared with serum osteocalcin concentrations before the injection of U266-MM cells (healthy) (*** p = 0.0005 and * p = 0.02, nonparametric Mann-Whitney t test). ( D ) Similarly, serum was analysed for carboxy-terminal collagen crosslinks (CTX1) levels by ELISA. Derived values were compared with serum CTX1 concentrations before the injection of U266-MM cells (healthy) (** p = 0.005 and ** p = 0.008 for healthy versus Tegavivint or combination, respectively, nonparametric Mann-Whitney t -tests).

Article Snippet: MM1R, NCIH929 and U266 Human Myeloma Cell Lines (HMCL) and HS5 (human fibroblasts) were obtained from the ATCC.

Techniques: In Vivo, Saline, Imaging, Micro-CT, Derivative Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Injection

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) mRNA and protein expression of S100A9 were measured in human primary macrophages after administration with normal glucose (NG; 5 mmol/L glucose) and high glucose (HG; 25 mmol/L glucose) by real-time quantitative polymerase chain reaction (qPCR) (left, administered for 6 hours, n = 16 PBMC donors) and Western blot (right, administered for 24 hours). These blots from one donor are representative of the 7 PBMC donors. The graph shows the signal intensity of S100A9 / β-actin ratio (n = 7 PBMC donors). (B) S100A9 levels in supernatants of human primary macrophages were measured by ELISA after administration with NG and HG condition for 24 hours (n = 17 PBMC donors). (C) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after stimulation with HG and recombinant human S100A9 (rhS100A9) for 6 hours (n=8 PBMC donors). (D) mRNA expression was measured in human primary macrophages after suppression by S100A9 siRNA for 48 hours and administration with HG for 6 hours (n = 6 PBMC donors). (E) mRNA and protein expression of RAGE were measured in human primary macrophages after stimulation with HG and rhS100A9 by real-time qPCR (left, stimulated for 6 hours, n = 8 PBMC donors) and western blot (right, stimulated for 24 hours). These blots from one donor are representative of the 4 PBMC donors. The graph shows the signal intensity of RAGE / β-actin ratio (n = 4 PBMC donors). (F) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 6 hours (n = 6 PBMC donors). P value was calculated by unpaired student’s t-test or one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) Size distribution and quantification of EVs derived from human primary macrophages were measured after stimulation with HG and rhS100A9 for 24 hours by nanoparticle tracking analysis (n = 9 PBMC donors). (B) Size distribution and quantification of EVs derived from human primary macrophages were measured after suppression by S100A9 siRNA for 48 hours and administration with NG and HG for 24 hours (n = 7 PBMC donors). (C) Size distribution and quantification of EVs derived from human primary macrophages were measured after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 24 hours (n = 7 PBMC donors). (D) The expression of S100A9 was measured within EVs derived from human primary macrophages by western blotting after stimulation with HG and rhS100A9 for 24 hours. These blots from one donor are representative of the 4 PBMC donors. (E) Calcific potential of EVs derived from human primary macrophages was measured by alkaline phosphatase (ALP) activity after stimulation with HG and rhS100A9 for 24 hours (n = 5 PBMC donors). (F) mRNA expression of the osteogenic factors, BMP2, BMP4, ALP, Runx2, osteopontin, and osteocalcin was measured in human primary macrophages after stimulation with HG and rhS100A9 for 12 hours (n = 8 PBMC donors). (G) mRNA expression of osteogenic factors was measured in human primary macrophages after suppression by S100A9 siRNA for 48 hours and administration with HG for 12 hours (n = 6 PBMC donors). (H) mRNA expression of osteogenic factors was measured in human primary macrophages after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 12 hours (n = 6 PBMC donors). P value was calculated by unpaired student’s t-test or one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Derivative Assay, Expressing, Western Blot, Activity Assay, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) THP-1 cells were cultured in either NG or HG, and then stimulated with rhS100A9 for the indicated times before being analyzed by Western blot with antibodies specific for the indicated targets. Data representative of the 3 different experiments. (B) Nrf2 in a nuclear extract of THP-1 cells (top panel, treated for 24 hours, 4 different experiments) was measured by Western blot and mRNA expression of detoxifying enzymes, hemeoxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (Nqo1) (bottom panel, treated for 6 hours) were measured after stimulation with HG and rhS100A9 (n = 4 PBMC donors). (C) The expression of Ser536 phosphorylation of p65 was measured in human primary macrophages by Western blot after pretreatment with VSC2 (10 μM) for 1 hour and stimulation with HG and rhS100A9 for 24 hours. The graph shows the signal intensity of phosphorylated p-65 / p65 ratio (n = 4 PBMC donors). (D) Nrf2 in a nuclear extract of THP-1 cells (top panel, treated for 24 hours, 3 different experiments) was measured by Western blot and mRNA expression of HO-1 and Nqo1 (bottom panel, treated for 6 hours, n = 4 PBMC donors) was measured after pretreatment with VSC2 (10 μM) for 1 hour and stimulation with HG and rhS100A9. (E) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 6 hours (n = 4 PBMC donors). (F) The number of EVs derived from human primary macrophages were measured after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 24 hours (n = 4 PBMC donors). (G) mRNA expression of osteogenic factors was measured in human primary macrophages after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 12 hours (n = 4 PBMC donors). (H) Calcific potential of EVs derived from THP-1 cells was measured by ALP activity after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 24 hours (3 different experiments). P value was calculated by one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Cell Culture, Western Blot, Expressing, Phospho-proteomics, Derivative Assay, Activity Assay, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: Apoe−/− mice fed a HCD for 18 weeks. After 10 weeks, streptozotocin (42 mg/kg/day) was intraperitoneally injected to diabetic group for 5 days, and fed a HCD for 8 weeks. (A) Von Kossa staining on longitudinal sections of the aortic arch; one of 14 animals per group is shown. Scale bars: 200 μm. The graph shows the percentage of positive area in the plaques. (B) Microcalcifications (yellow arrows) within the fibrous cap (white dotted line) were detected by Near-infrared fluorescent (NIRF) calcium tracer (OS680). The graph shows the number of microcalcifications (n=9). Scale bars: 50 μm. (C) Molecular imaging of aortic vascular calcification assessed by OsteoSense750 and vascular inflammation assessed by proteolytic activity, ProSense680. (n=6–10). The graph shows the SUM intensity related to total aortic area for fluorescent reflection imaging (FRI) in Apoe+/+S100a9+/+ (wild type; WT), Apoe−/− S100a9−/− (double knockout; DKO), and Apoe−/− S100a9+/+ (Apoe−/−) mice with diabetes and non-diabetic control. P value was calculated by paired student’s t-test or two-way ANOVA followed by Bonferroni test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Injection, Staining, Imaging, Activity Assay, Double Knockout, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) Macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9) were injected via tail vein, and F4/80 positive splenic macrophages were isolated. S100A9 mRNA was quantified by real-time PCR. (left panel, n = 3 per each group). Fluorescent in situ hybridization shows less S100A9 mRNA expression localized to macrophage-positive cells in Apoe−/− mice injected with macrophage-targeted LNP containing siS100a9. White arrows show the co-localization of S100A9 mRNA in macrophage-positive cells. One of 4 animals per group is shown (right panel). Scale bars: 20 μm. (B) mRNA expression of S100A9, inflammatory cytokines, and osteogenic factors was measured in splenic macrophages from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl (No diabetes and Diabetes) and siS100a9 (Diabetes with siS100a9) (n = 13, No diabetes and Diabetes with siS100a9; n = 16, Diabetes). (C) Immunohistochemical evaluation of ALP activity in aortic plaques from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 14, No diabetes and Diabetes with siControl; n = 13, No diabetes with siControl; n = 11, Diabetes with siS100a9). Scale bars: 100 μm. (D) In vivo molecular imaging of carotid arteries from diabetic Apoe−/− mice. Vascular calcification assessed by osteogenic activity, OsteoSense680 (red) and vascular inflammation assessed by proteolytic activity, ProSense750 (green). White arrow shows the co-localization of calcification and inflammation, indicating the formation of microcalcification (yellow). The image represents 7–10 mice per group. Scale bar = 200 μm. (E) Molecular imaging of aortic vascular calcification assessed by OsteoSense680 and vascular inflammation assessed by ProSense750. (n= 13–18). The graph shows the SUM intensity related to total aortic area for FRI in diabetic and non-diabetic Apoe−/− mice treated with macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9). (F) 3D micro-CT scanning of aorta from diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 4, per each group). The graph shows the mean number of microcalcifications assessed by size contribution. P value was calculated by paired student’s t-test or two-way ANOVA followed by Bonferroni test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Control, Injection, Isolation, Real-time Polymerase Chain Reaction, In Situ Hybridization, Expressing, Immunohistochemical staining, Activity Assay, In Vivo, Imaging, Micro-CT

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) 25 human atheroma obtained from carotid endarterectomy. Staining for S100A9, RAGE, and ALP in human atheroma (n=25). One of low S100A9 % positive area (left panel) and high S100A9 % positive area (right panel) are shown. Scale bars: 100 μm. Each graph shows the correlation between percentage of S100A9 positive area and ALP- and RAGE-positive area. The average of 5–10 high power fields was used for the analysis. (B) Serial section of human atheroma double-immune stained with anti-S100A9 (red) to anti-RAGE or anti-ALP (green) antibodies. Yellow area in Merge indicate S100A9 positive cells for either RAGE or ALP. Scale bars: 20 μm. The data represent 5 donors that showed similar results. (C) Transmission electron microscopy–based immunogold staining of S100A9 (red arrows) in EVs from human carotid plaques. One of 3 plaques is shown. Scale bar: 300 nm. (D) Carotid plaque specimens were obtained from diabetic (n = 4) and non-diabetic patients (n = 4) undergoing carotid endarterectomy. Volcano plot for the proteomics data. Red and blue markers indicate the significantly enriched (p<0.05) proteins in diabetic patient group and non-diabetic patient group, respectively, with a fold change (FC) cutoff of 2.0. (E) The schematic of the potential mechanism for macrophage-mediated EV microcalcification in diabetes. S100A9-RAGE axis may regulate pro-inflammatory and pro-osteogenic macrophage activation via Nrf2 and NF-κB pathways.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Staining, Transmission Assay, Electron Microscopy, Activation Assay

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Comparison of inflammatory-associated miRNA (inflamma-miRNA) profiles in SH- SWE and iNEU- PSEN cells, and in their respective exosomes, relatively to their SH- WT and iNEU- WT control samples. Cells were obtained and differentiated as described in Material and Methods. ( A ) Inflamma-miRNAs expression levels in both cellular AD models reveal that miR-124, miR-125b and miR-21 are increased. ( B ) Representative Transmission Electron Microscopy (TEM) images of exosomes with characteristic sizes after their isolation from cell secretomes by differential ultracentrifugation. ( C ) Representative Western blots for the common exosomal protein markers, Alix, CD63 and Flotilin-1 (FLOT-1). ( D ) Representative histograms of size and concentration of exosomes per sample. ( E ) Profiles of inflamma-miRNA cargo in exosomes from the different mutated cells evidencing increased expression levels of miR-124 and miR-155. Results are mean ± SEM fold change from at least three independent experiments (except NTA single data for exosomes from iNEU cell lines). * p < 0.05 and ** p < 0.01 vs. respective WT levels (dashed lines), two-tailed student’s t test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Comparison, Control, Expressing, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Concentration Assay, Two Tailed Test, Mutagenesis, Derivative Assay, Generated

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Expression levels of miR-124 in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells after transfection with the mock and with the miR-124 inhibitor and mimic. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Schematic representation of the miR-124 modulation with miR-124 inhibitor and/or mimic, as well as with scramble (respective negative controls—NC). ( B ) miR-124 expression levels before and after its modulation in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells. Results are mean ± SEM fold change from at least three independent experiments. ** p < 0.01 vs. respective mock control cells, one-way ANOVA with Bonferroni post-hoc test; # p < 0.05 and ## p < 0.01, between WT and AD mock controls, two-tailed student’s t -test. miR, miRNA; NC, negative control; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Expressing, Transfection, Control, Two Tailed Test, Negative Control, Mutagenesis, Derivative Assay, Generated

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Changes in dendrite outgrowth, mitochondria activation and fusion/fission dynamics by modulation of miR-124 with inhibitor and mimic, relatively to mock, in SH- WT and SH- SWE cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Representative fluorescence images of MAP-2 (green) for dendrites, Mitotracker (red) for mitochondria, and MFN-2 (green)/DRP-1 (red) for fusion/fission mitochondria dynamics. Cell nuclei are stained with Hoechst 33,258 dye (blue). Mitochondrial clumps are seen in the neurites (white arrowheads). ( B ) Evaluation of total dendrite length, primary dendrite length and primary dendrite number. ( C ) Quantification of fluorescence intensities for Mitotracker Red, DRP-1 and MFN-2. Results are mean ± SEM fold change, from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. # p < 0.05, between the same treatment in SH- WT and SH- SWE cells, two-tailed student’s t -test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1Δ E9 mutation; MAP-2, microtubule-associated protein 2; DRP1, protein dynamin-related protein 1; MFN-2, mitofusin-2 protein; FI, fluorescence intensity.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Activation Assay, Transfection, Fluorescence, Staining, Two Tailed Test, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Dendritic spine number decrease in iNEU- WT and iNEU- PSEN cells by modulation with the miR-124 inhibitor, but no changes were noticed for the mimic relatively to the mock transfection. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Representative fluorescence images of MAP-2 (green) staining in axons and dendrites in the different conditions. ( B ) Representative images of high magnification of dendritic segments (from panel A insets) with the miR-124 modulation. ( C ) Quantification of the dendritic spine number in each of the conditions. Results are mean ± SEM from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. miR, miRNA; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1 ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Transfection, Fluorescence, Staining, Derivative Assay, Generated, Control, Mutagenesis

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Effects of miR-124 modulation on APP transcription, Aβ accumulation and Tau phosphorylation in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Cellular quantification of APP gene expression by RT-qPCR revealing a decrease in the mutated cells with the miR-124 inhibitor. ( B ) Representative Western blots for Aβ species showing different representation and predominance of bands at 120 and 20 kDa. ( C ) Quantification of small and large Aβ oligomers showing that the first are increasingly represented in SH- SWE cells and the later in iNEU- PSEN cells. ( D ) Representative Western blots for Tau phosphorylation status at serine 404. ( E ) Quantification of phospho-Tau/total-Tau ratio revealing a decrease in iNEU-PSEN cells by the miR-124 mimic. Results are mean ± SEM fold change from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. # p < 0.05 and ## p < 0.05, between the same treatment in WT and mutated cells, two-tailed student’s t -test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Phospho-proteomics, Transfection, Gene Expression, Quantitative RT-PCR, Western Blot, Two Tailed Test, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: miR-124 is transmitted from donor to recipient neurons in a non-cell-contact co-culture system after transfection of miR-124 in SH- WT and SH- SWE donor cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods, before being co-cultured with non-transfected matched cells (recipient) for 24 h. ( A ) Schematic representation of SH- WT and SH- SWE donor cells transfected with mock and miR-124 mimic in co-culture with the matched non-transfected recipient cells. ( B ) miR-124 expression levels in the transfected and non-transfected cells after 24 h of co-incubation. Quantification was obtained by RT-qPCR and results are mean ± SEM fold change from at least three independent experiments. ** p < 0.01 vs. mock and # p < 0.05 vs. cells modulated with miR-124 mimic, two-tailed student’s t -test. miR, miRNA; SH-WT, human SH-SY5Y wild-type neurons; SH-SWE, human SH-SY5Y expressing the APP695 Swedish mutant protein.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Co-Culture Assay, Transfection, Cell Culture, Expressing, Incubation, Quantitative RT-PCR, Two Tailed Test, Mutagenesis

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Transfection of SH- WT /SH- SWE and of iNEU- WT /iNEU- PSEN cells with miR-124 inhibitor and miR-124 mimic influences miR-124 representation in the vesicle-free secretome (soluble) and packaging into exosomes, while also modifies the representation of other inflamma-miRNAs in the same cells and respective exosomes. ( A ) Evaluation of the influence of the cell treatment with miR-124 inhibitor and miR-124 mimic in the release of miR-124 into the secretome, as a soluble species and as a exosomal cargo. ( B ) Evaluation of cellular inflamma-miRNA expression levels after modulation of miR-124 with its mimic and inhibitor. ( C ) Evaluation of exosomal inflamma-miRNA expression levels after cellular modulation of miR-124 with its mimic and inhibitor. ( D ) Schematic representation of cellular and exosomal inflamma-miRNA distribution in the classical and in the advanced neuronal AD models by miR-124 inhibitor and miR-124 mimic. Results are mean ± SEM fold change from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Transfection, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: iScience

Article Title: Group 3 innate lymphocytes make a distinct contribution to type 17 immunity in bladder defence

doi: 10.1016/j.isci.2022.104660

Figure Lengend Snippet:

Article Snippet: RNeasy Micro kit , QIAGEN , Cat#74004.

Techniques: Virus, Recombinant, Protease Inhibitor, Control, Blocking Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Multiplex Assay, RNA Sequencing, Infection, Knock-Out, Software, Spectrophotometry, Microscopy

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 1 Mechanical force induces pyroptosis during OTM and alveolar bone remodeling in vivo. a Representative image of micro-CT and semiquantification analysis of force-induced OTM distance in rats. Scale bar: 1 mm. b Representative immunofluorescence images on the compression side of distobuccal roots and semiquantification analysis of double-labeled cells. Dashed lines mark the outline of distobuccal roots. Scale bar: 50 µm. c Representative tartrate-resistant acid phosphatase (TRAP) staining images of distobuccal roots. Scale bar: 200 µm. Results were presented as mean ± SD. n = 5 biologically independent samples. d Real time-PCR of Caspase-1, Il-1β, and Gsdmd in periodontal tissues. Results were presented as mean ± SD. n = 3 biologically independent samples. **P < 0.01, ***P < 0.001 versus Con; #P < 0.05, ##P < 0.01, ###P < 0.001 versus F3d; @P < 0.05, @@P < 0.01, @@@P < 0.001 versus F7d. The white arrow represents the direction of the force application. Large boxed areas show high magnification views of the small boxed areas

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: In Vivo, Micro-CT, Labeling, Staining, Real-time Polymerase Chain Reaction

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 2 Modulating pyroptosis level influences OTM and alveolar bone remodeling. a Schematic of the in vivo study. b Representative micro-CT images of OTM in mice. Con: mice without force stimuli in vivo; Force: mice receiving force stimuli for 7 d; Force+Polyphyllin VI (PPVI): mice receiving force stimuli for 7 d and the application of pyroptosis activator PPVI treatment. Force+MCC950: mice receiving force stimuli for 7 d and the application of pyroptosis inhibitor MCC950 treatment. Scale bar: 500 µm. c Representative immunohistochemical staining images and TRAP staining images of the compression side of distobuccal roots. The expressions of pyroptosis-related proteins Caspase-1, GSDMD, IL-1β, and the TRAP positive cells were detected. Large boxed areas show high magnification views of the small boxed areas. Scale bar: 100 µm. *P < 0.05, ***P < 0.001 versus Con; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Force; @@P < 0.01, @@@P < 0.001 versus Force+PPVI. Results were presented as mean ± SD. n = 5 biologically independent samples. The white arrow represents the direction of the force application

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: In Vivo, Micro-CT, Immunohistochemical staining, Staining

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 3 Force-induced pyroptosis modulates OTM and alveolar bone remodeling in a Caspase-1-depended manner. a Representative micro-CT images of force-induced tooth movement distance in wild type (WT) or Caspase-1−/−mice. Scale bar: 500 µm. b Representative immunohistochemical staining images and TRAP staining images of the compression side of distobuccal roots. Large boxed areas show high magnification views of the small boxed areas. Scale bars: 100 µm. **P < 0.01, ***P < 0.001 versus WT. c Representative images of micro-CT of force-induced tooth movement distance in mice with or without Caspase-1 inhibitor Belnacasan (VX765) application. Scale bar: 500 µm. d Representative immunohistochemically stained images and TRAP staining images of the compression sides of distobuccal roots. Large boxed areas show high magnification views of the small boxed areas. Scale bars: 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001 versus Con; #P < 0.05, ##P < 0.01 versus Force. The white arrow represents the direction of the force application. Results were presented as mean ± SD. n = 5 biologically independent samples

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Micro-CT, Immunohistochemical staining, Staining

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 4 Mechanical force induces pyroptosis in human PDL progenitor cells and influences osteoclastic activity. a Schematic of the isolation of ex vivo human PDL (h-PDL) progenitor cells. b Western blotting of pyroptosis-related proteins in ex-vivo h-PDL progenitor cells under clinical force for 7 d. c, d Western blotting and Real time-PCR of RANKL and OPG expressions in ex-vivo h-PDL progenitor cells. e ELISA of RANKL secretion in ex-vivo h-PDL progenitor cells. f Representative images of TRAP staining of osteoclasts in peripheral blood mononuclear cells (PBMCs) co-cultured with ex-vivo h-PDL progenitor cells. Scale bar: 50 µm. g Real time-PCR of TRAP and Cathepsin K (CTSK)expressions in PBMCs. *P < 0.05, **P < 0.01, ***P < 0.001 versus Con. h, i Western blotting of pyroptosis-related proteins in PDL progenitor cells under 1.5 g/ cm2 compressive force at different time points and under different force loading for 6 h in vitro. j Representative images of optical microscope (OM), scanning electron microscope (SEM) and transmission electron microscope (TEM) of PDL progenitor cells under 1.0 g/cm2 and 1.5 g/cm2 compressive force loading for 6 h in vitro. The red arrows in OM show the bubbles. Scale bar: 20 µm. The white circles in SEM show multiple pores in the membranes, and the red arrows in TEM show membrane disruption, cell swelling, and lysis. Scale bar: 1 µm. Results were presented as mean ± SD. n = 3–5 biologically independent samples

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Activity Assay, Isolation, Ex Vivo, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, In Vitro, Microscopy, Transmission Assay, Membrane, Disruption, Lysis

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 5 Regulation of PDL progenitor pyroptosis influences osteoclastic activity. a Western blotting of pyroptosis-related proteins in PDL progenitor cells under 1.5 g/cm2 force loading for 6 h with or without the appliance of PPVI and MCC950 in vitro. Con: PDL progenitor cells without force loading and drug application; Force: PDL progenitor cells under 1.5 g/cm2 force loading for 6 h; Force + PPVI: PDL progenitor cells with force loading and pyroptosis activator PPVI treatment. Force + MCC950: PDL progenitor cells with force loading and pyroptosis inhibitor MCC950 treatment. b Representative immunocytofluorescense images of PDL progenitor cells of Con, Force, Force + PPVI and Force + MCC950. Scale bar: 10 µm. n = 5 independent experiments. c Western blotting of RANKL and OPG in PDL progenitor cells of Con, Force, Force + PPVI and Force + MCC950. d Real time-PCR of RANKL, OPG, and the ratio of RANKL/OPG in PDL progenitor cells of Con, Force, Force + PPVI and Force + MCC950. e ELISA of RANKL secretion in PDL progenitor cells of Con, Force, Force + PPVI and Force + MCC950. *P < 0.05, **P < 0.01, ***P < 0.001 versus Con; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Force; @P < 0.05, @@P < 0.01, @@@P < 0.001 versus Force +PPVI. Results were presented as mean ± SD. n = 3–5 biologically independent samples

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Activity Assay, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 6 Regulation of Caspase-1 influences RANKL/OPG expression in PDL progenitor cells in vitro. a Western blotting of pyroptosis-related proteins in PDL progenitor cells under 1.5 g/cm2 force loading for 6 h with or without the appliance of the Caspase-1 inhibitor VX765. Con: PDL progenitor cells without force loading and drug application; Force: PDL progenitor cells under 1.5 g/cm2 force loading for 6 h; Force + VX765: PDL progenitor cells with force loading and VX765 treatment. b Representative immunocytofluorescense images of PDL progenitor cells under force loading with or without VX765 application. Scale bar:10 µm. n = 5 independent experiments. c Western blotting of RANKL and OPG in PDL progenitor cells under force loading with or without VX765 application. d Real time-PCR of RANKL, OPG, and the ratio of RANKL/OPG under 1.5 g/cm2 force loading for 6 h. e ELISA of RANKL secretion in PDL progenitor cells of Con, Force, Force+VX765. *P < 0.05, **P < 0.01, ***P < 0.001 versus Con; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Force. Results were presented as mean ± SD. n = 3–5 biologically independent samples

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Expressing, In Vitro, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 7 TRPV4 signaling is involved in force-induced pyroptosis in PDL progenitor cells. a Schematic of the experiment. b Western blotting of TRPV4 in ex-vivo h-PDL progenitor cells with or without clinical orthodontic force stimulated for 7 d. c Representative immunofluorescence images on the compression side of distobuccal roots. Dashed lines mark the outline of roots. Arrow represents the direction of the force. Scale bar: 50 µm. d Western blotting of pyroptosis-related proteins and semiquantification analysis in PDL progenitor cells under 1.5 g/cm2 force loading for 6 h. *P < 0.05, **P < 0.01, ***P < 0.001 versus Con, #P < 0.05, ##P < 0.01, ###P < 0.001 versus Force. e Representative immunofluorescence images of Fluo-4 (green) in PDL progenitor cells. Scale bar: 100 µm. Representative photomicrographs of intracellular reactive oxygen species (ROS) shown by H2DCF-DA (green) in PDL progenitor cells. Scale bar: 50 µm. Representative images of mitochondrial morphology detected by TEM (Scale bar: 1 µm). Representative images of mitochondrial morphology detected by MitoTracker Red (Scale bar: 5 µm). Representative images of PDL progenitor cells mitochondrial membrane potential detected by JC-1. Scale bar: 100 µm

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Western Blot, Ex Vivo, Membrane

Journal: International journal of oral science

Article Title: Force-induced Caspase-1-dependent pyroptosis regulates orthodontic tooth movement.

doi: 10.1038/s41368-023-00268-7

Figure Lengend Snippet: Fig. 8 Schematic showing force-induced Caspase-1-dependent pyroptosis in PDL progenitor cells via TRPV4 signaling, ultimately contributing to the activation of osteoclastogenesis and alveolar bone remodeling

Article Snippet: Mice were randomly divided into four groups as follows: (i) Force: force loading and 0.9% NaCl treatment every two days; (ii) Force+ PPVI: force loading and pyroptosis activator Polyphyllin VI (PPVI, S9302, Selleck) treatment (5 mg/kg every two days); (iii) Force+MCC950: force loading and pyroptosis inhibitor MCC950 (S7809, Selleck) treatment (20 mg/kg every two days); (iv) Control: the group without force loading and treatment.

Techniques: Activation Assay